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Load Dependent Assembly of the Bacterial Flagellar Motor

Load Dependent Assembly of the Bacterial Flagellar Motor

M any bacterial species swim, driven by transmembrane mo- lecular motors rotating extracellular flagellar filaments. In some species, for example, Salmonella, motor rotation is required both for swimming through liquid media (1) and for swarming across solid surfaces (2). Flagellar motors contain a series of rotat- ing protein rings (3). A schematic of the Escherichia coli flagellar motor can be seen in Fig. 1a. The C-ring, also called the switch complex, is made of 26 or more copies of FliG, 34 to 35 of FliM, and ~140 of FliN. The C-ring connects via the transmembrane rod to the extracellular flagellar filament and interacts with cell wall- anchored transmembrane stator complexes formed by the pro- teins MotA and MotB. E. coli motors are bidirectional: motors can switch between counterclockwise (CCW) and clockwise (CW) ro- tation in response to chemosensory signals, with switching trans- mitted through C-ring proteins FliM and FliN on the cytoplasmic side of FliG.

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A New Player at the Flagellar Motor: FliL Controls both Motor Output and Bias

A New Player at the Flagellar Motor: FliL Controls both Motor Output and Bias

Flagellated bacteria are sensitive to a variety of environmental factors and are well known to use a chemotaxis system to transmit information about their chemical environment to the flagellar motor (2). Conversely, they use the flagellar apparatus to transmit information about the physical environment to various transcrip- tional and posttranscriptional pathways that ensure an appropri- ate developmental response. For example, swimming in viscous solutions or on surfaces triggers changes such as induction of lat- eral flagellar synthesis in Vibrio parahaemolyticus (45, 46) or up- regulation of flagellar and virulence genes in Proteus mirabilis, responses that enhance swarming (47). In the former case, changes in lateral flagellar gene expression also occur by exposure to sodium channel blockers, indicating that they might be caused by interference with the function of the sodium ion-powered sta- tors that drive the polar flagellum (48). In C. crescentus, surface adherence via retractile pili, which restricts rotation of the flagel- lum, induces secretion of holdfasts that attach bacteria to the sur- face (49). In Bacillus subtilis, inactivation of the stators induces the transcription of genes for the synthesis of a highly mucoid poly- mer that promotes biofilms (50, 51). While the flagellar motor is clearly implicated as a sensory device in all these bacteria, how such a device would work is not yet understood. It is likely that external mechanical stimuli and internal disengagement of stators (52), whether they alter proton flux, stator occupancy, or switch conformation, ultimately all manipulate the rotor-stator inter- face, relaying the signal to rotor- or stator-associated proteins. FliL, which interacts with both rotor and stator, may provide an important handle to probe this sensory device.

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Structure of Vibrio FliL, a New Stomatin like Protein That Assists the Bacterial Flagellar Motor Function

Structure of Vibrio FliL, a New Stomatin like Protein That Assists the Bacterial Flagellar Motor Function

The motor torque is generated by electrostatic interaction between FliG and the stator A-subunit (11). The stator B-subunit interacts with the peptidoglycan layer via the periplasmic region to anchor the stator unit (12, 13). The periplasmic region of the B-subunit contains a plug region that regulates the ion influx through the stator (14). The stator units are not static but dynamically assemble into and disassemble from the motor (11, 15). Although each single stator unit generates enough torque to rotate the filament under low-load conditions, a number of stators assemble around the rotor under high-load conditions to generate sufficient torque, suggesting that the flagellar motor somehow senses the load and adjusts its torque to the load by changing the number of stators around the rotor (1–3, 16, 17). A recent mutational study showed that the long cytoplasmic loop region of the A-subunit is responsible for the load- dependent assembly of the stator unit in Salmonella (18).

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Diversification of Campylobacter jejuni Flagellar C Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes

Diversification of Campylobacter jejuni Flagellar C Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes

Flagellar motor C rings do not all follow this compositional blueprint. For example, Bacillus subtilis and Thermotoga maritima encode FliY in place of FliN (51–53). The domain organization of FliY is similar to that of FliM, with a phosphatase domain of the CheC/CheX/CheY family and a SPOA domain (53, 54). While FliN and FliY are mutually exclusive in most bacteria, Leptospira species and Epsilonproteobacteria, including Campylobacter and Helicobacter species, produce both FliN and FliY, although FliY in these bacteria lacks active sites for phosphatase activity (55, 56). An initial study in Helicobacter pylori indicated that FliN and FliY are required for full flagellation and motility (56, 57). Structural and interaction studies demonstrated that FliY interacts with FliM and FliN separately to assemble a three-protein complex (57). Furthermore, H. pylori FliY-FliN and FliY-FliM heterodimers interact with FliH in vitro, but how these proteins contribute to C-ring composition and architecture in H. pylori remains un- known (57). The presence of both FliY and FliN SPOA-containing proteins in Epsilon- proteobacteria prompts questions regarding the function and location of these pro- teins, how have they diverged relative to an ancestral FliN-like protein, and what selective benefits drove retention of two FliN/FliY-like proteins.

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Stoichiometry and Turnover of the Bacterial Flagellar Switch Protein FliN

Stoichiometry and Turnover of the Bacterial Flagellar Switch Protein FliN

ABSTRACT Some proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evi- dence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direc- tion of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, la- beled with the fluorescent protein CyPet, in Escherichia coli. Our results confirm that, in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ⴞ 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ⴞ 17 and 144 ⴞ 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by aver- aged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments.

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Mutations in the Borrelia burgdorferi Flagellar Type III Secretion System Genes fliH and fliI Profoundly Affect Spirochete Flagellar Assembly, Morphology, Motility, Structure, and Cell Division

Mutations in the Borrelia burgdorferi Flagellar Type III Secretion System Genes fliH and fliI Profoundly Affect Spirochete Flagellar Assembly, Morphology, Motility, Structure, and Cell Division

Disruption of either fliH or fliI had profound effects on B. burg- dorferi cellular morphology, motility, flagellar motor and filament structure, and cell division. At the ultrastructural level, cryo-ET revealed that inactivation of either fliH or fliI resulted in the loss of densities associated with both protein products, indicating that the presence of both proteins is required for the stable formation of an FliH-FliI complex at the cytoplasmic apex of the secretion apparatus. Surprisingly, the absence of either FliH or FliI did not prevent assembly of the remainder of the flagellar motor, although the number of motors did decrease from the WT level of approx- imately 8 to about 6 per cell pole. Flagellar filaments were also present on most motors, although with decreased lengths; the fil- aments appeared to be truncated at the hook level in approxi- mately 10% of the motors. In other bacteria, the ATPase FliI facil- itates the delivery of export substrates to the secretory apparatus, while FliH is involved in the positioning of FliI near the export channel (33, 44, 46, 53). Our results indicate that, in B. burgdorferi, FliH and FliI are not required for uptake of protein substrates by the export apparatus; thus, their absence does not prevent the formation of flagellar motors and filaments. However, export of the FlaB filament protein is much less efficient, resulting in shorter filaments. These results are comparable to those obtained by Er- hardt et al. with Salmonella Typhimurium (41). In their studies, flagellar filament assembly and swimming motility were minimal

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Novel Components of the Flagellar System in Epsilonproteobacteria

Novel Components of the Flagellar System in Epsilonproteobacteria

We found that CJJ81176_0413 interacts with PflA, a protein exclusively encoded by Epsilonproteobacteria (65) and previously shown to be required for motility (46). Interestingly, disruption of pflA results in apparently normal but paralyzed flagella (46), the same phenotype observed after the disruption of CJJ81176_0413. Electron microscopy examination of the C. jejuni ⌬CJJ81176_0413 mutant showed apparently normal flagella at both poles (Fig. 4) although the mutant was completely defective in motility (Fig. 3). These observations suggest a role for these proteins in the function and/or assembly of the flagellar motor. Despite a lack of detectable primary amino acid sequence similar- ity between CJJ81176_0413 and PflA, structural homology searches indicated that these proteins share structural similarities to the same O-linked N-acetylglucosaminyltransferase (PDB 1W3B [66]), suggesting that these two proteins are structurally similar. C. jejuni flagellin and other flagellar components are known to be glycosylated, a modification that is required for fla- gellar assembly (31, 67–69). It is possible that CJJ81176_0413 as well as PflA may be involved in this process. Intriguingly, both these proteins have tetratricopeptide (TPR) repeats, which previ- ous studies have implicated in conferring substrate specificity to eukaryotic N-acetylglucosaminyltransferases (70). The presence of these repeats in CJJ81176_0413 as well as PflA is consistent with a potential role in protein glycosylation. Interestingly, CJJ81176_0413 also interacted with KdpD and CJJ81176_1442, the latter of which belong to a cluster of genes implicated in the biosynthesis of the capsular polysaccharide of C. jejuni (71), sug-

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Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

Comparative proteomic analysis revealed that alde- hyde-alcohol dehydrogenase enzyme (CAP0035), 3- hydroxybutyryl-CoA dehydrogenase enzyme (CAC2708), flagellar motor switch protein - FliY (CAC2215) which controls the swimming of the bacterium, and flagellin family hook associated protein (CAC2203) were highly up-regulated in C. acetobutylicum from glucose utilized ABE fermentation than xylose. In addition, the NSAF values of the enzymes acetoacetyl-coenzyme A: acetate/ butyrate coenzyme A-transferase (CoA-transferase) and butyraldehyde dehydrogenase (BAD) were found to be relatively abundant only on the glucose substrate. These results are consistent with the literature which demon- strate that a highly motile inoculum results in higher solvent production and non-motility leads to no solvent due to the loss of CoA-transferase and BAD enzymes that are directly involved in solvent production [57,58]. Recent transcriptomic studies of C. acetobutylicum growing on mixtures of glucose and xylose also reported that genes for chemotaxis proteins and flagellin biosynthesis are activated through glucose [59]. There- fore, the mechanism that C. acetobutylicum uses for both glucose and xylose sugar resulting in a preference for glucose [60] could also be attributed to the high motility nature of C. acetobutylicum grown on glucose compared to less motility with xylose substrate. On the other hand, the expression of rare lipoprotein-A (CAP0058) was found to be highly up-regulated in

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Functional roles of the transverse and longitudinal flagella in the
swimming motility of Prorocentrum minimum (Dinophyceae)

Functional roles of the transverse and longitudinal flagella in the swimming motility of Prorocentrum minimum (Dinophyceae)

In conclusion, we propose the functions of the two flagella of P. minimum are as follows: the transverse flagellum acts as a propulsion device, to move the cell along the longitudinal axis of the helical swimming path and rotate it about its antero-posterior axis; the longitudinal flagellum acts as a rudder, to produce a helical swimming trajectory, and controls the orientation of the cell. Flagellar hairs on the transverse flagellum are probably present because they are necessary to produce simulated cell motion, in agreement with that observed in P. minimum. This is the first numerical evaluation of the functions of the transverse and longitudinal flagella of a dinoflagellate.

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Flagellar Elongation and Gene Expression in Chlamydomonas reinhardtii

Flagellar Elongation and Gene Expression in Chlamydomonas reinhardtii

Lithium (Li ⴙ ) affects the physiology of cells from a broad range of organisms including plants and both vertebrate and invertebrate animals. Although its effects result presumably from changes in gene expression elicited by its interaction with intracellular signal transduction pathways, the molecular mechanisms of Li ⴙ action are not well understood. The biflagellate green alga Chlamydomonas reinhardtii is an ideal genetic model for the integration of the effects on Li ⴙ on signal transduction, gene expression, and aspects of flagellar biogenesis. Li ⴙ causes C. reinhardtii flagella to elongate to ⬃ 1.4 times their normal length and blocks flagellar motility (S. Nakamura, H. Tabino, and M. K. Kojima, Cell Struct. Funct. 12:369–374, 1987). We report here that Li ⴙ treatment increases the abundance of several flagellar mRNAs, including ␣ - and ␤ -tubulin and pcf3-21. Li ⴙ -induced flagellar gene expression occurs in cells pretreated with cycloheximide, suggesting that the abundance change is a response that does not require new protein synthesis. Deletion analysis of the flagellar ␣ 1-tubulin gene promoter showed that sequences necessary for Li ⴙ -induced expression differed from those for acid shock induction and contain a consensus binding site for CREB/ATF and AP-1 transcription factors. These studies suggest potential promoter elements, candidate factors, and signal transduction path- ways that may coordinate the C. reinhardtii cellular response to Li ⴙ .

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Protein Kinase Involved in Flagellar-Length Control

Protein Kinase Involved in Flagellar-Length Control

While most of these mutant axonemes had the canonical 9-plus-2 microtubule structure, 11% lacked the central doublet (Fig. 7I and J), which was never observed in the wild type. As some of the 9-plus-0 flagella were found to be separate from all cell bodies (Fig. 7J), the sections were not located in the flagellar pocket and therefore did not represent the transition zone, a region from which the central microtubule doublet is absent (Fig. 7K). The 9-plus-0 structures were also found in flagella attached to the cytoplasmic membrane of the null mutant, displaying tight junction-like structures indicating the proximity to the opening of the flagellar pocket (Fig. 7I). How- ever, most of the transverse sections displaying flagella in their pockets revealed the 9-plus-2 microtubule pattern (Fig. 7M) even in the situation of a dividing cell with the old and new flagella side by side (data not shown). The remaining 21.5% of the flagellar transverse sections revealed a rudimentary PFR in the mutant compared with wild-type PFR (Fig. 7E to H). FIG. 4. Southern analysis of genomic DNA. (A) A total of 5 ␮g of

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Rab23 is a flagellar protein in Trypanosoma brucei

Rab23 is a flagellar protein in Trypanosoma brucei

Given the increasing evidence for flagellar-associated functions for mammalian Rab23 and the evolutionary correlation between Rab23 and motile flagella, we sought to re-examine the location of TbRab23. Re-inves- tigation of both the original antisera and a new anti- peptide sera suggested significant issues with specificity (data not shown and Additional file 2). For example, we observed high molecular weight cross-reacting bands in blots using the original antisera, which may represent detection of highly repetitive polypeptides at the nuclear envelope [39], and the peptide sera did not recognise endogenous TbRab23 by Western blot or IF. Given the low level of expression of TbRab23 as a confounding factor and questionable specificity of antisera, we chose an independent strategy to re-examine TbRab23 location.

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Flagellar synchronization through direct hydrodynamic interactions

Flagellar synchronization through direct hydrodynamic interactions

The origin of flagellar synchronization has been the subject of intense theoretical investigation for many decades. One of the earliest experimental results was Rothschild's qualitative observation (Rothschild, 1949) that the flagella of bull spermatozoa tend to synchronize when they swim close to one another, coupled only through the fluid surrounding them. Much more recent observations of self- organised vortex arrays of swimming sea urchin spermatazoa near surfaces (Riedel et al., 2005) provide further evidence for synchrony mediated purely by hydrodynamic coupling. Motivated by Rothschild's observation, Taylor (Taylor, 1951) developed a mathematical model in which two laterally infinite, inextensible sheets with prescribed sinusoidal travelling waves of transverse deformation interact with each other through a viscous fluid. He found that the rate of viscous dissipation is mini- mised when the two sheets are in phase. While minimisation of dissipation often holds in real phys- ical systems, it is not in general a fundamental principle from which to deduce dynamical processes. Rather, an explanation for synchronization should capture the forces and torques associated with *For correspondence:

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Antiphase synchronization in a flagellar dominance mutant of Chlamydomonas

Antiphase synchronization in a flagellar dominance mutant of Chlamydomonas

There are four key observations. First is the existence of the AP state itself [Fig. 1(d)], visualized by discrete wave- forms within one cycle, color coded in time, and overlaid on a spatial map of average flagellar residence time. Compare this to Fig. 1(a) showing the wt IP breaststroke. Here, the flagella simultaneously execute extended ‘‘power strokes’’ followed by high-curvature ‘‘recovery strokes,’’ in which they are drawn forward with distal portions sliding past the body. In the AP of ptx1, distinct power and recovery strokes are clearly discernible, but as one flagellum executes the former, the other proceeds through the latter. The mutant also displays an IP state [Fig. 1(c)] that is nearly [12] identical to the wt IP. For example, the areas A wt;ptx1 IP swept out by the flagellum in both cases (i.e., the areas within residence-time plots in Fig. 1) agree to within 1%. In the case of ptx1, evident also is the drastic reduction in spatial extent spanned by both flagella during AP relative to the wt IP mode. This alteration of beating waveform occurs with an abrupt increase in beating fre- quency, which together comprise our second observation. We extract flagellar phases c cis;trans from Poincare´ section- ing of the dynamics [5] and define the interflagellar phase difference as ¼ ð c trans c cis Þ= 2 . For a typical ptx1 cell, Fig. 2(a) tracks ðtÞ over 40 s as it fluctuates around half-integer values during AP, but around integer values during IP. As seen in Fig. 2, our third finding is that flagella of ptx1 stochastically transition between IP and AP modes, in a manner reminiscent of the synchronous or asynchro- nous transitions of the wt [5]. Figure 2(b) shows that the instantaneous beat frequency is indeed higher in AP ( AP : 82 4 Hz) than in IP ( IP : 58 5 Hz). Fourth, we

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Flagellar Localization of a Helicobacter pylori Autotransporter Protein

Flagellar Localization of a Helicobacter pylori Autotransporter Protein

Unlike ImaA and VlpC, which are localized to a bacterial pole, FaaA is localized to the flagella. Correspondingly, faaA mutants exhibit multiple flagellar abnormalities, including absence of fla- gella, decreased numbers of flagella, increased flagellar fragility, and mislocalization of flagella to the lateral side of the bacteria instead of the pole. In addition, faaA mutant bacteria exhibit de- creased motility compared to the WT strain. Thus, FaaA is re- quired not only for flagellar stability and proper flagellar localiza- tion but also for optimal flagellar function. In an analysis of gastric colonization, a faaA mutant strain colonized the mouse stomach less efficiently than WT bacteria at an early time point postinfec- tion, which is consistent with a known essential role of motility at early stages of H. pylori infection (36). FaaA might also have a role at later stages of infection, since flagella are likely to be required for continuous H. pylori colonization of the gastric mucus layer dur- ing the natural turnover of gastric mucus and exfoliation of gastric epithelial cells.

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Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

Isolation of flagella from Chlamydomonas reinhardtii and characterization of their phosphoproteins. For identification of phosphorylated flagellar proteins of C. reinhardtii, we first isolated flagella from vegetative cells, with deflagellation by the dibucaine method (55). Flagella were then purified by sucrose cushions and demembranized by Nonidet NP-40 treatment in the presence of phosphatase inhibitors (see Materials and Methods). The resulting MMA fraction was first analyzed by immunodetection to find out if contaminating proteins were present. For this purpose, antibodies directed against the chlo- roplastic vesicle-inducing protein in plastids 1 (VIPP1) (17) and against the cytosolic C1 subunit of the RNA-binding pro- tein CHLAMY1 (63) were used. While these antibodies de- tected VIPP1 and C1 in a protein crude extract, they did not show any reaction with proteins from the MMA fraction (Fig. 1A and B). Thus, contaminations of the MMA fraction with proteins of these major subcellular compartments, and proba- bly also of others, should be minor. As a positive control, an antibody against CK1, which is enriched in flagella in compar- ison to a crude extract (41), was used (Fig. 1C). It showed a strong signal in the MMA fraction. A comparison of proteins from a crude extract and proteins from the MMA fraction by Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) corroborated the enrichment of specific proteins (Fig. 1D).

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Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies

Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies

Flagellar length synchronization narrows the steady-state flagellar length distribution. Synchronous culture provides a better way to address cell cycle and related flagellar dynamics. However, cell cycle synchronization methods provide only partial synchronization and thus show high variance in flagellar length. To obtain 100% flagellar length (F-L) synchronization, we exploited an inherent property of Chlamy- domonas, which is their ability to regenerate the flagella after amputation (38). We first performed different synchronization methods and then compared their steady-state flagellar lengths to those of nonsynchronized cells (Fig. 1a, Fig. S1 in the supplemental material, and Table S1 in the supplemental material). For F-L synchronization, we tested different regeneration time durations following deflagellation to determine the time point at which flagellar length variability is minimized. We found that the predeflag- ellation flagellar length distribution was broad, which was expected as our starting culture was nonsynchronous and contained a heterogeneous population of cells (Fig. 1b, red). After deflagellation, all flagella started to grow synchronously but the length distribution still remained broad at 2 and 2.5 h, when some cells were still in the 8- to 9- ␮ m size range and did not reach their original length (Fig. 1b, light green). The distribution narrowed and became maximally homogeneous at 3 h (Fig. 1b, medium green) (Table S2). However, the length distribution remained narrow for only a short time, expanding again within 30 min and increasing with time (Fig. 1b, dark

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A Regulatory Checkpoint during Flagellar Biogenesis in Campylobacter jejuni Initiates Signal Transduction To Activate Transcription of Flagellar Genes

A Regulatory Checkpoint during Flagellar Biogenesis in Campylobacter jejuni Initiates Signal Transduction To Activate Transcription of Flagellar Genes

transduction, we examined whether production of these proteins was affected in different C. jejuni flagellar mutants. Immunoblot analysis of C. jejuni lysates revealed wild-type levels of FliF and FliG in FlgSR TCS or flagellar T3SS mutants (Fig. 2A). We also detected wild-type levels of the FlgS histidine kinase and FlgR response regulator in mutants lacking FliF, FliG, and T3SS pro- teins (Fig. 2C). In both assays, the levels of RpoA (the ␣ subunit of RNAP) were similar in all strains, verifying that similar amounts of proteins from lysates were analyzed (Fig. 2A and C). Due to small amounts of flagellar T3SS proteins in C. jejuni, it was diffi- cult to assess their abundance. However, we were able to monitor FlhB production (Fig. 2D). FlhB undergoes autoproteolytic cleav- age during flagellar biogenesis (10, 21). We detected similar levels of processed FlhB in the wild-type strain and mutants lacking FlgSR, FliF, or FliG (Fig. 2D). These data indicated that the flagel- lar proteins required for signal transduction via the FlgSR TCS were produced independently of each other, yet alone they were not sufficient to initiate signaling. Instead, FliF, FliG, and the fla- gellar T3SS are all required to activate ␴ 54 -dependent flagellar

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CMF22 Is a Broadly Conserved Axonemal Protein and Is Required for Propulsive Motility in Trypanosoma brucei

CMF22 Is a Broadly Conserved Axonemal Protein and Is Required for Propulsive Motility in Trypanosoma brucei

CMF22 knockdown completely blocks propulsive cell motility but does not cause flagellar paralysis. Rather, flagella with the CMF22 knockdown exhibit an abnormal beating pattern charac- terized by erratic beating and frequent reversals of waveform propagation. The organism with the knockdown does not show any indication of defective flagellum assembly or major defects in growth that accompany gross disruptions of axoneme structure in T. brucei (23, 64–67). Indeed, TEM analysis did not reveal any obvious defect in axoneme ultrastructure. These data argue for a role of CMF22 in flagellar beat regulation, rather than a structural role or a direct role in generating forces that power microtubule FIG 9 CMF22-HA is less stably associated with the axoneme in NDRC mutants. (A) Western blot of the indicated subcellular fractions from trypanin-UTR- knockdown, CMF70-knockdown, or CMF22-ORF-knockdown cells probed with anti-HA, antitrypanin, or antitubulin antibody, as indicated. Fractions corre- spond to whole-cell lysate (lanes L), nonionic detergent-soluble (lanes S1) and insoluble (lanes P1) fractions, as well as soluble (lanes S2) and insoluble (lanes P2) fractions obtained by extraction of P1 with 1 M NaCl. The lower-molecular-mass bands in the anti-HA and antitrypanin blots are likely degradation products.

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Schlesner, Matthias
  

(2008):


	The halobacterium salinarum taxis signal transduction network: a protein-protein interaction study.


Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Schlesner, Matthias (2008): The halobacterium salinarum taxis signal transduction network: a protein-protein interaction study. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

The role of OE2404R remained unclear. The ∆4 strains were not distinguishable from wildtype strains in the phototaxis measurement and with respect to the flagellar rotational bias but produced significantly smaller swarm rings. Reduced swarm ring size is generally considered as outcome of diminished chemotaxis capability (given that the motility in itself is not affected). Several alternative hypotheses can be envisioned to explain the differences in the phototaxis measurements (no difference to wt) and swarming (reduced when compared to wt). First, OE2404R might only be involved in chemotaxis and not phototaxis signalling. Second, this protein might be required for fast and effective adaptation. Third, OE2404R might be required for fine tuning of the response. The first hypothesis seems rather unlikely since no other evidence exists that chemical and light signals utilise separate pathways in H. salinarum. The other possibilities are related to the phototaxis assay: This assay monitors the reaction after one strong and sudden change in light intensity, but does not report the adaptation efficiency or the reaction to more subtle stimuli. Further experiments should be done to test these three hypotheses. Dose-response curves for the phototactic behaviour would be a promising approach to discriminate between hypothesis one and three. Monitoring the cellular response after repeated phototactic stimulation could be used to test hypothesis two.

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